Association of Common Variants in OLA1 Gene with Preclinical Atherosclerosis

Abstract

Reactive oxygen species impair the blood vessels, leading to the initiation of atherosclerosis, and migration and proliferation of vascular smooth muscle cells and neovascularization by endothelial cells of vasa vasorum are essential for atherosclerosis development. Obg-like ATPase 1 (OLA1), a negative regulator in cellular responses to oxidative stress, binds to breast cancer susceptibility gene 1 (BRCA1), which protects vascular endothelial and smooth muscle cells against reactive oxygen species. However, it is not known whether OLA1 is genetically correlated with atherosclerosis. Here, we conducted two independent population-based case-control studies to explore the effects of variants in OLA1 genes on preclinical atherosclerosis. A total of 564 and 746 subjects who had thicker and normal carotid intima-media thickness (cIMT), respectively, were enrolled. Among 55 screened SNPs, rs35145102, rs201641962, rs12466587, rs4131583, and rs16862482 in OLA1 showed significant associations with cIMT. SNP rs35145102 is a 3'-utr variant and correlates with the differential expression of OLA1 in immune cells. These five genetic markers form a single closely linked block and H1-ATTGT and H2-GCCTC were the top two most prevalent 5-locus haplotypes. The H1 + H1 genotype negatively and H1 + H2 genotype positively correlated with thicker cIMT. The five identified SNPs in the OLA1 gene showed significant correlations with cIMT. Furthermore, we found that OLA1 was required for migration and proliferation of human aortic endothelial and smooth muscle cells and regulated vascular tube formation by human aortic endothelial cells. Therefore, these genetic variants in the OLA1 gene may serve as markers for risk prediction of atherosclerotic diseases.

Keywords: BRCA1; OLA1; atherosclerosis; carotid intima–media thickness; genetic association study; population-based study; single nucleotide polymorphism.

Figure 1

Schematic illustration of approaches to determine genetic association of OLA1, BRCA1, and BARD1 with cIMT.

Figure 2

LD map of the five OLA1 candidate SNPs (values are r²).

Figure 3

Effect of OLA1 RNAi (RNA interference) on proliferation and migration of human aortic SMCs. (A,B) Depletion of OLA1 in human aortic SMCs. After human aortic SMCs were transfected with non-silencing (NS) or OLA1 siRNA duplexes for 42 h, cells were collected and lysed for immunoblot analysis with antibodies against OLA1 and GAPDH (loading control). Immunoblot images of both OLA1 and GAPDH are shown in (A). The band intensities of OLA1 and GAPDH from (A) were determined using LS software from VisionWorks (UVP, LLC). The band intensity of OLA1 was normalized to that of GAPDH, and the relative protein levels of OLA1 normalized to the non-silencing treatment in the graph shown in (B) are the means ± s.d. (error bars) from three independent experiments. ***, p < 5 × 10⁻⁷. (C) Depletion of OLA1 causes a reduced proliferation of human aortic SMCs. After human aortic SMCs were transfected with the indicated siRNA duplexes for 42 h, 1.5 × 10⁴ cells were incubated with the BrdU-containing medium for 8 h. The BrdU-incorporated cells were stained using 3,3′-diaminobenzidine tetrahydrochloride for analysis (see the Materials and Methods section (Section 4.8)). Each independent experiment was quadruplicated. In the graphs, values are the relative percentages of the amounts of incorporated BrdU in cells normalized to the non-silencing treatment and the means ± s.d. (error bars) from three independent experiments. ***, p ˂ 5 × 10⁻⁴ (Student’s t-test). (D) Depletion of OLA1 causes reduced migration ability in human aortic SMCs. After human aortic SMCs were transfected with the indicated siRNA duplexes for 42 h, 3 × 10⁴ cells were resuspended in 100 μL of 0.5% FBS medium for the trans-well assay (see the Materials and Methods section (Section 4.8)). The cells stuck in the pores of the filter were imaged and quantitated. Each independent experiment was triplicated. In the graphs, values are the relative percentages of migrating cells normalized to the non-silencing treatment and the means ± s.d. (error bars) from three independent experiments. **, p ˂ 5 × 10⁻⁵ (Student’s t-test).

Figure 4

Effect of OLA1 RNAi on proliferation and migration of and tube formation by human aortic ECs. (A,B) Depletion of OLA1 in human aortic ECs. After human aortic ECs were transfected with non-silencing (NS) or OLA1 siRNA duplexes for 42 h, cells were collected and lysed for immunoblot analysis with antibodies against OLA1 and GAPDH (loading control). Immunoblot images of both OLA1 and GAPDH are shown in (A). The band intensities of OLA1 and GAPDH from (A) were determined using LS software from VisionWorks (UVP, LLC). The band intensity of OLA1 was normalized to that of GAPDH, and the relative protein levels of OLA1 normalized to the non-silencing treatment in the graph show in (B) are the means ± s.d. (error bars) from four independent experiments. **, p < 5 × 10⁻⁵. (C) Depletion of OLA1 causes a reduced proliferation of human aortic ECs. After human aortic ECs were transfected with the indicated siRNA duplexes for 42 h, 1.5 × 10⁴ cells were incubated with the BrdU-containing medium for 8 h. The BrdU-incorporated cells were stained using 3,3′-diaminobenzidine tetrahydrochloride for analysis. Each independent experiment was duplicated. In the graph, values are the relative percentages of the amounts of incorporated BrdU in cells normalized to the non-silencing treatment and the means ± s.d. (error bars) from three independent experiments. *, p ˂ 5 × 10⁻⁴ (Student’s t-test). (D) Depletion of OLA1 causes reduced migration ability in human aortic ECs. After human aortic ECs were transfected with the indicated siRNA duplexes for 42 h, 3 × 10⁴ cells were resuspended in 100 μL of 0.5% FBS medium for the trans-well assay. The cells stuck in the pores of the filter were imaged and quantitated. Each independent experiment was triplicated. In the graphs, values are the relative percentages of migrating cells normalized to the non-silencing treatment and the means ± s.d. (error bars) from three independent experiments. **, p ˂ 5 × 10⁻⁵ (Student’s t-test). (E,F) Depletion of OLA1 causes reduced capillary tube formation by human aortic ECs. After human aortic ECs were transfected with the indicated siRNA duplexes for 42 h, 5 × 10⁴ ECs were used for Matrigel tube formation assay (see the Materials and Methods section (Section 4.8)). The formed capillary tubes were imaged as shown in (E) and quantitated as shown in (F). Each independent experiment was triplicated. In the graph shown in (F), values are the relative percentages of the formed capillary tubes normalized to the non-silencing treatment and the means ± s.d. (error bars) from three independent experiments. ***, p ˂ 1 × 10⁻⁶ (Student’s t-test).